The reaction of hemoglobin with its ligands has been used as a model protein reaction for many years. In spite of the fact that more is known about this protein than any other biological molecule, the most important point, i.e. the physical and chemical processes that occur upon reaction with oxygen are not yet understood. Deland has constructed a comprehensive thermodyanmic model of hemoglobin, and Ackers has developed a thermodynamic model of the dimer-tetramer equilibrium which includes the regulatory cooperative structural changes. The Ackers model is general in nature and deals with internal regulation of the rate of chemical reaction by proposing changes in the conformation of the protein itself. Crucial to the testing of these theories is accurate data on the oxygen binding curve and on the intermediates formed during the oxygenation process. To provide this information we have completed a new oxygen equilibrium curve apparatlus which will be used at the CDC to obtain the new binding data on a variety of mutant hemoglobins. A second instrument, called a cryo-quench flow, since the chemical reaction is stopped by quickly (50 milliseconds) lowering the temperature to -30 degrees C, has been built and tested using the determination of the kinetic intermediates of carbon monoxide binding to hemoglobin. The results of this work show that there is never a measureable amount of triply liganded hemoglobin formed because the process moves so quickly to a state in which four carbon monoxide molecules are bound. A new inertial pulsed flow system has been designed and built to lower the quench time to .5 milliseconds so that the intermediates formed with oxygen could be studied. Quench times of 15 milliseconds have been reached, and with modifications we should soon achieve the shorter time. More importantly, only .25 ml of each reagent is needed. This instrument will be a replacement for our highly successful chemical quench flow system which now requires 2.5 ml per reaction. This instrument is presently being used by ten major laboratories throughout the world for Na, K. ATPASE and Ca - Sacroplasmic reticulum studies.